Magnesium ions perform many essential roles in all living organisms, including enzymatic catalysis, structural integrity, and microbial pathogenesis; however, the mechanisms that regulate homeostasis are incompletely understood. Proteins are generally assumed to be the sentinels for metal ions; however, our central hypothesis is that a highly conserved metal-sensing RNA is broadly used among bacteria for posttranscriptional control of magnesium homeostasis. Given the biological distribution of this RNA element it is likely to constitute a common mode of metal ion regulation and its discovery suggests the presence of other RNA-based metal ion sensors. We have demonstrated that this RNA functions as a genetic sensor for magnesium in Bacillus subtilis, the model system for Gram-positive bacteria, and controls expression of one of the three magnesium transporter classes. Together, these data suggest that association of magnesium provokes a conformational switch within the RNA that in turn stabilizes a transcription termination signal for reduction of downstream gene expression. Based on these observations we will elucidate the mechanism of magnesium recognition by the RNA-based sensor and investigate the individual contribution of the regulatory RNA to overall magnesium homeostasis control. The Specific Aims are to: 1). Elucidate the structural basis for metal ion detection. We will complete the three-dimensional resolution of the metal-bound RNA complex. Using additional biophysical and biochemical methods we will explore the specificity and stoichiometry of RNA-magnesium interactions, which we will also functionally test through specific substitution of RNA groups that directly coordinate to magnesium. 2). We will study the dynamical nature of metal-induced RNA folding by gel- and fluorescence-based techniques in relation to terminator formation. We will also investigate whether discontinuous transcriptional elongation is specifically coupled with these other processes as a final requirement for regulation. 3). We expect that this RNA-mediated mechanism, although vitally important, may only be one of multiple genetic mechanisms required for magnesium homeostasis. We will test this hypothesis by investigating posttranscriptional regulation of the candidate transport genes within the context of additional magnesium- regulated genes. [unreadable] [unreadable] [unreadable]